We previously showed for dengue type 4 (DEN4) that both NS2B and NS3 together constitute a protease that cleaves the NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions. Others have proposed that NS3 is a viral protease based on the finding that the N-terminal 180 amino acids of NS3 contains four regions of homology to serine proteases. The role of NS2B in the protease activity is less clear. We initiated studies to mutagenize NS2B and the protease domain of NS3 to further characterize their roles in proteolytic processing. The PTM 1/v TF7 transient expression system was employed to study proteolytic processing of NS2B-30%NS3. Efficient cleavage yielding NS2B and 30%NS3 was observed in cells transfected with recombinant DNA coding for polyprotein precursor. Seven mutants were produced by introducing a deletion into the NS2B portion of this construct. Five of these mutants cleaved as efficiently as the wild-type sequence. One mutant showed slightly reduced cleavage, while another mutant ( Bgl II/Nru I) was completely defective for cleavage. Further mutagenesis in this important region should identify the residues of NS2B critical for protease activity. Expression of NS2B-30%NS3 in E. coli from the IPTG inducible tac promoter was lethal to cells. Incorporation of the Bgl II/Nru I mutation into this construct did not eliminate this lethality, whereas truncation of NS3 allowed growth. This observation is consistent with the notion that expression of NS3 alone, which contains the proposed protease domain, is lethal to E. coli. If this is the case, it should be possible to isolate mutants that are defective for the NS3 protease activity.